ripk3 inhibitor gsk  (MedChemExpress)

Journal: Inflammation

Article Title: ZBP1 Mediates Renal Tubular Injury in Diabetic Nephropathy Through RIPK3-mediated Necroptosis

doi: 10.1007/s10753-025-02362-w

Figure Lengend Snippet: ZBP1 expression in renal tissue is associated with tubular necroptosis in patients with DN. (a) The IHC of RIPK3 and MLKL in each group. Scale bars = 50 μm. (b) Immunofluorescence double staining of p-MLKL and AQP1. Scale bars = 50 μm. (c) We investigated transmission electron micrographs from NC group ( n = 5), DN1 group ( n = 3), and DN2 group ( n = 7). The images depicted in the figure show electron micrographs of DN2 group patients. TEM observed thickened glomerular basement membranes, some renal tubular atrophy, and swollen proximal tubular epithelial cells in the DN group (red puncta). Abbreviations: IHC, immunohistochemistry; NC, normal control; DN1, diabetic nephropathy patients with interstitial fibrosis and tubular atrophy score < 2; DN2, diabetic nephropathy patients with interstitial fibrosis and tubular atrophy score ≥ 2; AQP, aquaporin. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Additionally, MTECs were treated with the RIPK3 inhibitor GSK’872 (MCE, HY-101872) for 24 h for further study.

Techniques: Expressing, Immunofluorescence, Double Staining, Transmission Assay, Immunohistochemistry, Control

Journal: Inflammation

Article Title: ZBP1 Mediates Renal Tubular Injury in Diabetic Nephropathy Through RIPK3-mediated Necroptosis

doi: 10.1007/s10753-025-02362-w

Figure Lengend Snippet: ZBP1 knockdown inhibits RIPK3-mediated necroptosis in db/db mice. (a) IHC assay for RIPK3 and MLKL protein expression on tubules in db/db mice. Scale bars = 50 μm. (b) IF double staining for p-MLKL and AQP1 in the db/db kidney. Scale bars = 50 μm. (c) RIPK3, p-RIPK3, MLKL and p-MLKL levels were assessed by Western blot. (d) TEM showed swollen endoplasmic reticulum and mitochondria (red puncta) in the proximal renal tubular epithelial cells, as well as loss of mitochondrial cristae in db/db mice compared to NC group, whereas the ZBP1 knockdown group exhibited alleviated lesions. Abbreviations: IHC, immunohistochemistry; EV, empty vector; KD, knockdown; IF, immunofluorescence; TEM, transmission electron microscopy. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Additionally, MTECs were treated with the RIPK3 inhibitor GSK’872 (MCE, HY-101872) for 24 h for further study.

Techniques: Knockdown, Expressing, Double Staining, Western Blot, Immunohistochemistry, Plasmid Preparation, Immunofluorescence, Transmission Assay, Electron Microscopy

Journal: Inflammation

Article Title: ZBP1 Mediates Renal Tubular Injury in Diabetic Nephropathy Through RIPK3-mediated Necroptosis

doi: 10.1007/s10753-025-02362-w

Figure Lengend Snippet: ZBP1 knockdown reduces HG-induced MTECs damage and necroptosi s in vitro. (a and b) The knockdown effect of ZBP1 was detected by real-time PCR and Western blot. (c) Western blot analysis of KIM1 in MTECs. (d) IF of KIM1 in MTECs. ZBP1 knockdown reduced KIM-1 expression in HG-induced MTECs. Scale bars = 50 μm. (e) Quantitative data and Western blotting of RIPK3, p-RIPK3, MLKL and p-MLKL. (f) Immunofluorescence analysis of p-MLKL in MTECs. Scale bars = 50 μm. (g) TEM revealed that HG increased MTEC volume, induced cell membrane rupture (blue puncta), cytoplasmic translucency (yellow puncta), organelle swelling (red puncta) and cytoplasmic content leakage. These pathological changes were attenuated in the ZBP1 knockdown group. Abbreviations: EV, empty vector; KD, knockdown; IHC, immunohistochemistry; HG, high glucose; IF, immunofluorescence; TEM, transmission electron microscopy. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Additionally, MTECs were treated with the RIPK3 inhibitor GSK’872 (MCE, HY-101872) for 24 h for further study.

Techniques: Knockdown, In Vitro, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunofluorescence, Membrane, Plasmid Preparation, Immunohistochemistry, Transmission Assay, Electron Microscopy

Journal: Inflammation

Article Title: ZBP1 Mediates Renal Tubular Injury in Diabetic Nephropathy Through RIPK3-mediated Necroptosis

doi: 10.1007/s10753-025-02362-w

Figure Lengend Snippet: ZBP1 knockdown attenuates AGEs-induced MTECs damage and necroptosis in vitro. (a and b) Western blot and real-time PCR of KIM-1 in MTECs. (c) Quantitative data and Western blotting of RIPK3, p-RIPK3, MLKL and p-MLKL. (d) Immunofluorescence analysis of p-MLKL in MTECs. Scale bars = 25 μm. (e) Real-time PCR analysis mRNA levels of IL-1β and TNF-α. (f) Quantitative data and Western blot analysis of p-p65 in MTECs stimulated with AGEs. (g) Western blot showed that inhibition of ZBP1 expression could inhibit the expression of α-SMA and COL-I. Abbreviations: AGEs, advanced glycation end-products; EV, empty vector; KD, knockdown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Additionally, MTECs were treated with the RIPK3 inhibitor GSK’872 (MCE, HY-101872) for 24 h for further study.

Techniques: Knockdown, In Vitro, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence, Inhibition, Expressing, Plasmid Preparation

Journal: Inflammation

Article Title: ZBP1 Mediates Renal Tubular Injury in Diabetic Nephropathy Through RIPK3-mediated Necroptosis

doi: 10.1007/s10753-025-02362-w

Figure Lengend Snippet: ZBP1 interacts with RIPK3 in HG-induced MTECs. ( a ) Western blot showed the protein levels of RIPK3, p-RIPK3, MLKL and p-MLKL. ( b ) Immunofluorescence images showed that ZBP1 knockdown and GSK’872 reduced the phosphorylation and membrane translocation of MLKL in HG-induced MTECs. ( c ) Western blot revealed that overexpressing RIPK3 negated the protective effects of ZBP1 knockdown on necroptosis. ( d - f ) Co-IP, SPR and molecular docking of ZBP1 and RIPK3. ( e ) Molecular docking of ZBP1 and RIPK3. Docking scale = 6.88 Kcal/mol. Abbreviations: EV, empty vector; KD, knockdown; HG, high glucose; IF, immunofluorescence; IP, immunoprecipitation; SPR, surface plasmon resonance. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Additionally, MTECs were treated with the RIPK3 inhibitor GSK’872 (MCE, HY-101872) for 24 h for further study.

Techniques: Western Blot, Immunofluorescence, Knockdown, Phospho-proteomics, Membrane, Translocation Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Immunoprecipitation, SPR Assay

Journal: Science Advances

Article Title: Reprogramming RIPK3-induced cell death in malignant B cells promotes immune-mediated tumor control

doi: 10.1126/sciadv.adv0871

Figure Lengend Snippet: ( A ) Expression of Casp8, FADD, RIPK1, RIPK3, and MLKL in whole-cell extracts from pro-B cell tumors by Western blot. MEFs and BMDMs were used as a positive control. Data are representative of three independent experiments. ( B ) Protein quantification from (A) expressed as a fold change over MEF expression. Results are compiled from three independent experiments. ( C ) MLKL expression from bulk RNA-seq (Cancer Cell Line Encyclopedia database) performed on 19 human B cell lines originating from ALL, Burkitt lymphoma, and DLBCL. THP-1 cells were used as positive control. ( D ) MLKL expression from bulk RNA-seq (ImmGen database) in different stages of murine B cell development. Peritoneal macrophages and endothelial cells were used as positive controls. GC, germinal center. ( E ) MLKL protein expression assessed on murine splenic B cells, pro-B tumors, and MEFs. ( F ) t-SNE representation of single-cell RNA-seq (10X Genomics public datasets) from frozen human BM mononuclear cells. B cell development stages are highlighted. ( G ) Violin plots of MLKL expression in various stages of human B cell development from (F). Myeloid cells were used as positive control. ( H ) Chromatin accessibility of the MLKL locus in mouse primary cells was assessed by ATACseq. The gray area highlights changes in ATACseq peaks within the TSS and exon 1. Wilcoxon rank sum test was used for statistical analysis (G). *** P < 0.001.

Article Snippet: In some conditions, cells were pretreated for 24 hours with recombinant mouse IFN-β (2.5 ng/ml; BioLegend) before being cultured with the B/B dimerizer in the presence or absence of zVAD-fmk and RIPK3 kinase inhibitor GSK′843 (5 μM; MedChemExpress).

Techniques: Expressing, Western Blot, Positive Control, RNA Sequencing

Journal: Science Advances

Article Title: Reprogramming RIPK3-induced cell death in malignant B cells promotes immune-mediated tumor control

doi: 10.1126/sciadv.adv0871

Figure Lengend Snippet: ( A ) Expression by Western blot of Casp8, RIPK1, RIPK3, and MLKL in whole-cell extracts from pro-B cell tumors pretreated with type I or type II IFNs. Data are representative of two independent experiments. MEF cells were used as positive control. ( B ) Kinetics of cell death in actRIPK3-pro-B cells treated or not with IFN-β for 24 hours and cultured later with B/B dim in the presence or absence of zVAD-fmk. ( C ) Representative plots showing the staining act-Casp3/LD in actRIPK3-pro-B cells stimulated for 3 hours with B/B dim with or without zVAD-fmk and IFN-β pretreatment. Numbers indicate the percentage of gated cells. ( D ) Frequency of cells expressing act-Casp3 and/or incorporating LD from (C). ( E and F ) actRIPK3-pro-B cells expressing or lacking MLKL were cultured with B/B dim with or without zVAD-fmk and IFN-β pretreatment. Cell death was assessed by flow cytometry. (E) Representative plots and (F) quantification. Results in [(C) to (F)] are representative of three independent experiments. Data are expressed as mean ± SEM. ( G ) Time-lapse images showing morphological changes in actRIPK3-pro-B cells pretreated with IFN-β and exposed to B/B dim with or without zVAD-fmk. Scale bars, 10 μm. Data are representative of three independent experiments. ( H ) Scheme summarizing the type and magnitude of cell death induced by RIPK3 signaling in pro-B tumors in the presence or absence of caspase inhibition and IFN-β.

Article Snippet: In some conditions, cells were pretreated for 24 hours with recombinant mouse IFN-β (2.5 ng/ml; BioLegend) before being cultured with the B/B dimerizer in the presence or absence of zVAD-fmk and RIPK3 kinase inhibitor GSK′843 (5 μM; MedChemExpress).

Techniques: Expressing, Western Blot, Positive Control, Cell Culture, Staining, Flow Cytometry, Inhibition

Journal: Science Advances

Article Title: Reprogramming RIPK3-induced cell death in malignant B cells promotes immune-mediated tumor control

doi: 10.1126/sciadv.adv0871

Figure Lengend Snippet: ( A to C ) Pro-B cells expressing or lacking endogenous RIPK3 pretreated (or not) with IFN-β were cultured in the presence or absence of SM BV6 and pan-caspase inhibitor emricasan (Emri). (A and B) Frequency of cell death without (A) or with (B) IFN-β pretreatment. (C) Frequency of cells expressing act-Casp3 and/or incorporating LD dye. ( D and E ) WT mice bearing pro-B tumors were treated with three doses of IFN-β and three doses of SM birinapant (Biri) with or without Emri. (D) Experimental design. (E) Frequency of tumor cells among CD45 + cells in the blood, lymph nodes (LNs), and BM 14 days after tumor inoculation. Each dot represents one mouse. Data are representative of three independent experiments with n = 5 mice per group in each experiment. ip, intraperitoneal. ( F ) Survival of WT or Rag2 −/− B6 mice harboring pro-B cell tumors and treated with a combination of birinapant, emricasan, and IFN-β. n = 12 to 14 mice per group compiled from two independent experiments. ( G and H ) Induction of tumor-specific CD8 + T cell responses upon cell death reprograming therapy. (H) H-2K b -OVA tetramer staining was performed 14 day post–pro-B–OVA cell injection in mice treated with the indicated drug combination ( n = 4 mice per group). Representative of two independent experiments. Data are expressed as mean ± SEM. One-way ANOVA was used for statistical analysis [(A), (B), (E), and (H)]. ** P < 0.01, and *** P < 0.001.

Article Snippet: In some conditions, cells were pretreated for 24 hours with recombinant mouse IFN-β (2.5 ng/ml; BioLegend) before being cultured with the B/B dimerizer in the presence or absence of zVAD-fmk and RIPK3 kinase inhibitor GSK′843 (5 μM; MedChemExpress).

Techniques: Expressing, Cell Culture, Staining, Injection